Figure 1. Engineering Essential Amino Acid (EAA) biosynthesis in metazoan cells.

(A) Presence of amino acid biosynthesis pathways across representative diverse organisms on Earth. (B) Schematic of EAA biosynthesis pathway steps that require engineering in mammalian cells to enable complete amino acid prototrophy if imported from Escherichia coli. Proline and Valine pathways shown in this work are highlighted in red. (C) Workflow diagram of a synthetic genomics approach involving pathway design, construction, integration and testing towards mammalian EAA restoration.

Figure 1—figure supplement 1. Amino acid dropout growth assays in CHO-K1.

(A) Growth assays on Chinese hamster ovary (CHO)-K1 cells seeded into media with reduced or omitted essential amino acids (Figure 1—figure supplement 1—source data 1). Percentages represent the relative media amino acid concentration compared to standard F-12K medium. 100% corresponds to 0.02 mM (Trp), 0.05 mM (Tyr), 0.06 mM (Ile, Met, Phe), 0.2 mM (Ala, Asn, Asp, Glu, Gly, Leu, Ser, Thr, Val), 0.22 mM (His), 0.4 mM (Cys, Lys), 0.6 mM (Pro), and 2 mM (Arg, Gln), respectively. Error bars show SD of three replicates. (B) CHO-K1 cells grow with minimal defects in F-12K medium lacking glycine and alanine, but are more sensitive to arginine starvation (Figure 1—figure supplement 1—source data 1). (C) CHO cells do not grow without exogenous proline, confirming the known epigenetic proline auxotrophy found in this cell line (Figure 1—figure supplement 1—source data 1). Error bars represent data from three replicates.

Figure 1—figure supplement 2. Imported bacterial genes rescue CHO epigenetic proline auxotrophy.

(A) Proline biosynthetic pathway from glutamate. (B) Schematic representation of two constructs to test bacterial gene rescue of Chinese hamster ovary (CHO)-K1 auxotrophy, one expressing the E. coli proA and proB genes, encoding glutamate-5-semialdehyde dehydrogenase and glutamate-5-kinase, respectively (pPro), and a control construct expressing only mCherry (pCtrl-mCh). Constructs are shown as they appear following integration into the Flp-In CHO line. (C) Hoechst 33348 live nuclei microscopy shows cells expressing the pPro construct grow in the absence of proline and appear healthy. Scale bar represents 50 μm and is shared across images (D) Growth assay of cells seeded into proline-free F-12K medium and grown for 5 days (Figure 1—figure supplement 2—source data 1). The bacterial gene pPro pathway resulted in robust growth recovery in proline-free medium. Error bars represent data from three replicates.